Hygiena KIT 2300 48 User manual

Brand: Hygiena

Model: KIT 2300 48

Type: User manual

Hygiena KIT 2300 48 is a real-time PCR kit for the qualitative detection of Listeria monocytogenes DNA in food and environmental samples. It includes reagents for up to 96 reactions and is compatible with various real-time PCR instruments. The kit targets a specific gene found in all subgroups of Listeria monocytogenes, ensuring high inclusivity. It also features an internal amplification control to monitor PCR performance. The detection limit is as low as 1 to 10 cells per 25 g sample, depending on the sample preparation method.

Documentation for the qualitative detection of

Listeria monocytogenes DNA

Listeria

monocytogenes

Detection Kit

MANUAL

foodproof®

Order No. KIT 2300 48

Kit for 96 reactions for a

maximum of 94 samples

Store kit at -25 °C to -15 °C

For testing of food

and environmental samples

Manual:

Version 5, February 2022

Listeria monocytogenes

Detection Kit

foodproof®

Approvals:

LICENSE NUMBER 070401

KIT 2300 48 - Listeria monocytogenes Detection Kit

TABLE OF CONTENTS

1. OVERVIEW ..................................................................................4

1.1 General Information ................................................................................ 4

1.2 Applicability .............................................................................................. 4

1.3 Kit Contents ............................................................................................. 5

2. INSTRUCTIONS ..........................................................................6

2.1 Required Material .................................................................................... 6

2.2 Precautions and Preparations ................................................................ 7

2.3 Enrichment and DNA extraction ............................................................ 8

2.3.1 Certiﬁed Methods ................................................................................................ 8

2.4 Procedure ................................................................................................. 9

2.4.1 Workﬂow .............................................................................................................. 9

2.4.2 Program Setup ...................................................................................................10

2.4.3 Data Interpretation ............................................................................................. 10

2.5 Troubleshooting ..................................................................................... 11

2.6 Support ................................................................................................... 12

3. ADDITIONAL INFORMATION ................................................... 13

3.1 Testing Principle .................................................................................... 13

3.2 Trademarks ............................................................................................ 14

3.3 Reference Number ................................................................................ 14

3.4 Change Index ......................................................................................... 14

KIT 2300 48 - Listeria monocytogenes Detection Kit

OVERVIEW

1. OVERVIEW

1.1 General Information

Number of Reactions

The kit is designed for 96 reactions with a ﬁnal reaction volume of 25 µl each. Up to 94

samples plus positive and negative control can be analyzed per run.

Storage and Stability

Store all components at -25 °C to -15 °C. They are guaranteed to be stable through the

expiration date printed on the label. Opening of the kit does not shorten the expiration date.

1.2 Applicability

The kit described in this manual has been developed for real-time PCR instruments

with a FAM and a VIC/HEX detection channel. The performance of the kit was tested

with the following real-time PCR instruments: LightCycler® 480, LightCycler® 96 (Roche

Diagnostics), Applied BiosystemsTM 7500 Fast (Thermo Scientiﬁc), Mx3005P® (Agilent),

and others.

The foodproof® Listeria monocytogenes Master Mix is sequence-speciﬁc for a mpl-

gene found in all subgroups of Listeria monocytogenes. Inclusivity has been tested with

102 Listeria monocytogenes isolates whereas all of them could be detected (100 %

inclusivity). Exclusivity was determined using 60 non-Listeria monocytogenes bacteria.

A relative detection limit of 1 to 10 cells per 25 g sample can be achieved with all kinds

of foods. The foodproof® Listeria monocytogenes Detection Kit detects down to 103

- 104 cfu/ml in enrichment cultures, depending on the sample preparation kit used:

foodproof® StarPrep Two Kit or foodproof® ShortPrep II Kit.

KIT 2300 48 - Listeria monocytogenes Detection Kit

OVERVIEW

Component Details

1Master Mix

(yellow cap)

3 x 600 µl, ready-to-use primer and hydrolysis probe mix speciﬁ c for

parameter DNA and the parameter-speciﬁ c Internal Control (IC).

Store at -25 °C to -15 °C.

Avoid repeated freezing and thawing! Protect from light!

2Enzyme Solution

(red cap)

3 x 32 µl, contains Taq DNA Polymerase and Uracil-DNA Glycosylase

(heat labile) for prevention of carry-over contamination.

Store at -25 °C to -15 °C.

3Internal Control

(white cap)

3 x 32 µl, contains a stabilized solution of plasmid DNA and a yellow dye

for better visualization. For use as an internal ampliﬁ cation control.

Store at -25 °C to -15 °C.

Optional: After ﬁ rst thawing store at 2 °C to 8 °C for up to one month.

4Control Template

(purple cap)

1 x 50 µl, contains a stabilized solution of DNA. For use as a PCR run

positive control.

Store at -25 °C to -15 °C.

Optional: After ﬁ rst thawing store at 2 °C to 8 °C for up to one month.

5Negative Control

(transparent cap)

1 x 1 ml, contains PCR-grade water. For use as a PCR run negative control.

Store at -25 °C to -15 °C.

Optional: After ﬁ rst thawing store at 2 °C to 8 °C for up to one month.

KIT 2300 48

1.3 Kit Contents

A schematic representation of the foodproof® Listeria monocytogenes Detection Kit with

all its components.

KIT 2300 48 - Listeria monocytogenes Detection Kit

INSTRUCTIONS

2. INSTRUCTIONS

2.1 Required Material

Most of the required equipment and reagents are available through HygienaTM.

Please contact us for further information.

Real-time PCR compatible strips or plates with optical cap

or foil

 Sterile reaction tubes for preparing PCR mixes and dilutions

Material

Nuclease-free, aerosol-resistant pipette ﬁlter tips

Use a real-time PCR cycler suitable for detection of respective probes.

PCR strip or plate centrifuge

KIT 2300 48 - Listeria monocytogenes Detection Kit

INSTRUCTIONS

2.2 Precautions and Preparations

The kit provides all reagents required for the PCR. However, in order to achieve reliable

results, the entire assay procedure must be performed under nuclease-free conditions.

Follow the instructions below to avoid nuclease, carry-over, or cross-contamination:



Keep the kit components separate from other reagents in the laboratory.



Use nuclease-free labware (e.g., pipettes, pipette tips, reaction vials).



Wear gloves when performing the assay.



To avoid cross-contamination of samples and reagents, use fresh aerosol barrier

pipette tips.

To avoid carry-over contamination, transfer the required solutions for one experiment

into a fresh tube, rather than directly pipetting from stock solutions.



Physically separate the workplaces for DNA preparation, PCR setup, and PCR to

minimize the risk of carry-over contamination. Use a PCR hood for all pipetting steps.



Sample Material: Use any sample material suitable for PCR in terms of purity,

concentration, and absence of inhibitors.



DNA Extraction: We provide sample preparation kits suitable for all kind of food

samples and primary production stage samples.



Positive Control: Always run a positive control with the samples. Use the provided

control DNA (Control Template) or a positive sample preparation control.



Negative Control: Always run a negative control with the samples. To prepare

a negative control, replace the template DNA with PCR-grade water. Include a

negative control during sample preparation to monitor reaction purity and cross-

contamination. This extraction control can be used as an additional negative control

reaction.



Conﬁ rmation

: If required, positive results may be conﬁ rmed by appropriate methods

(e.g., reference method).



Waste Disposal:

All contaminated and potentially infectious material, like enrichment

cultures or food samples, should be autoclaved before disposal and eliminated

according to local rules and regulations. For more information, e.g., proper disposal

of unused chemicals, please refer to the appropriate safety data sheet (SDS).

For more information, please refer to the appropriate safety data sheet (SDS). The SDS is

available online at www.bc-diagnostics.com.

Keep the PCR mix away from light.

KIT 2300 48 - Listeria monocytogenes Detection Kit

INSTRUCTIONS

2.3 Enrichment and DNA extraction

The foodproof® Listeria monocytogenes Detection Kit is intended for the rapid detection

of Listeria monocytogenes DNA isolated from enrichment cultures prepared by valid

methods and inoculated with all kinds of foods that are potentially contaminated with

Listeria monocytogenes. The detection kit must not be used in diagnostic procedures.

Pre-enrichment broth and temperature according to ISO 11290 or BAM (Chapter 10) or

USDA for 24 to 48 h. Other suitable, validated enrichment procedures can also be used.

Recommended DNA extraction kits:

ÖKIT 2301 77 - StarPrep Two (suitable for most matrices)

ÖKIT 2301 71 - ShortPrep II Kit (for a very quick extraction)

2.3.1 Certiﬁed Methods

This AOAC-RI validated kit is based on the foodproof® Listeria monocytogenes Detection

Kit - Hybridization Probes (LightCycler® 1.x, 2.0) which has been AOAC RI and NordVal

validated.

KIT 2300 48 - Listeria monocytogenes Detection Kit

INSTRUCTIONS

2.4.1 Workﬂow

Thaw the solutions, mix by ﬂicking the tubes four to ﬁve times, and brieﬂy spin vials in a

microcentrifuge before opening.

2.4 Procedure

This protocol describes how to perform the analysis of DNA extracts by real-time PCR.

3. ADD SAMPLES AND CONTROLS

Pipette 5 µl of samples, negative control (colorless cap) or

Control Template (purple cap) into respective wells.

2. ADD PCR MIX

Pipette 20 µl of prepared PCR mix into each strip or plate well.

1. PREPARE PCR MIX

Add 18 µl of Master Mix (yellow cap), 1 µl Enzyme Solution (red cap) and

1 µl Internal Control (white cap) for each reaction to a suitable tube

(n samples + 2 controls + at least one additional reaction to cover pipetting loss).

Mix carefully but thoroughly by pipetting up and down.

PREPARATION OF THE PCR MIX

Take appropriate precautions to prevent contamination, e.g. by using ﬁ lter tips and wearing gloves.

Thaw reagents, mix (do not vortex!), and brieﬂ y spin vials before opening.

4. SEAL

Seal strips/plate accurately.

5. CENTRIFUGE

Brieﬂ y spin strips/plate in a suitable centrifuge.

6. START REAL-TIME PCR RUN

Cycle samples as described in the program setup (2.4.2).

+20 µl

+5 µl

+18 µl + 1 µl + 1 µl

KIT 2300 48 - Listeria monocytogenes Detection Kit

INSTRUCTIONS

2.4.2 Program Setup

Program your real-time PCR instrument before setting up the PCR reactions. Select the

following channels:

u FAM (L. monocytogenes) and VIC (Internal Control).

As an alternative to VIC, HEX can be used. For the PikoReal® 24, Yakima Yellow has to be

selected.

For some real-time PCR instruments the probe quencher as well as the usage of a passive

reference dye has to be speciﬁed. This kit contains probes with TAMRA as quencher and no

passive reference dye.

For users of the Agilent Mx3005P instrument: Click “Instrument” and “Filter Set Gain

Settings” to open the Filter Set Gain Settings dialog box in which the gain settings may be

viewed and modiﬁed. For FAM the Filter Set Gain Setting has to be modiﬁed to “x1”.

2.4.3 Data Interpretation

Verify results of positive (Control Template) and negative controls (H2O), before interpreting

sample results. Always compare samples to positive and negative control. Review data from

each channel and interpret results as described in the table.

FAM VIC Result Interpretation

+ + or - Positive for L. monocytogenes

- + Negative for L. monocytogenes

- - Invalid

Pre-incubation: 1 cycle

Step 1: 37 °C for 4 min

Step 2: 95 °C for 5 min

Ampliﬁ cation: 50 cycles

Step 1 : 95 °C for 5 sec

Step 2*: 60 °C for 60 sec

* Fluorescence detection

1 cycle

4 min 5 min 5 sec | 60 sec*

50 cycles

95 °C

60 °C

37 °C

KIT 2300 48 - Listeria monocytogenes Detection Kit

INSTRUCTIONS

2.5 Troubleshooting

Problem Possible Cause Recommendation

No signal

increase is

observed, even

with positive

controls.

Incorrect detection channel has

been chosen.

Set channel settings for respective dyes

accordingly.

Pipetting errors. Check for correct reaction setup and repeat

the PCR run.

Always run a positive control along with

your samples.

No data acquisition programmed. Check the cycle programs.

A sample shows

no signals,

including the

internal control.

Positive and

negative control

have proper

signals.

Inhibitory effects of the sample

material (e.g., caused by

insufficient purification).

Use the recommended DNA extraction kit.

Dilute samples or pipette a lower amount of

sample DNA (e.g., 20 µl PCR-grade water

and 5 µl sample instead of 25 µl sample).

Negative control

samples are

positive.

Carry-over contamination. Exchange all critical solutions and reagents

for DNA/RNA extraction.

Repeat the complete experiment with fresh

batches of all reagents.

Always handle samples, kit components

and consumables in accordance with

commonly accepted practices to prevent

carry-over contamination.

Add positive controls after sample and

negative control reaction vessels have been

sealed.

Fluorescence

intensity is too

low.

Inappropriate storage of kit

components.

Store lyophilized PCR mix at 2 °C to 8 °C,

protected from light and moisture.

Low initial amount of target DNA. If possible, increase the amount of sample

DNA. Depending on the chosen DNA isolation

method, inhibitory effects may occur.

Reagents are not homogeneously

mixed.

Mix reagents thoroughly before pipetting.

Fluorescence

intensity varies.

Insufficient centrifugation of the

PCR strips, e.g., resuspended

PCR mix is still in the upper part of

the vessel or bubbles trapped in

the mix.

Always centrifuge PCR strips.

Use the centrifuge models and settings

recommended in this manual.

Avoid the introduction of air bubbles during

pipetting.

Outer surface of the vessel or the

seal is dirty (e.g., by direct skin

contact).

Always wear gloves when handling the

vessels and seal.

Do not mark vessels on the outside of the

tubes or directly on top of the reaction mix.

KIT 2300 48 - Listeria monocytogenes Detection Kit

INSTRUCTIONS

2.6 Support

If you have questions or experience any problems with our products, please contact us:

www.hygiena.com/technical-support-request

Our aim is to provide you with a solution as quickly and effectively as possible. We would

also like you to contact us if you have any suggestions for improving the product or in case

you would like to use our product for a different application. We highly value your feedback.

KIT 2300 48 - Listeria monocytogenes Detection Kit

ADDITIONAL INFORMATION

3.1 Testing Principle

3. ADDITIONAL INFORMATION

The foodproof® kit provides all necessary reagents and a control template for reliable

interpretations of results. To ensure maximum reliability of the kit and to prevent

misinterpretation of negative results due to inhibition of the ampliﬁ cation, an Internal

Control (IC) is included. A hydrolysis probe was designed to bind speciﬁ cally the IC,

allowing detection in the respective channel, whereas the target DNA is detected in another

channel. In case of a negative result due to inhibition of the ampliﬁ cation by the sample

DNA of interest, the ampliﬁ cation of the IC is suppressed as well, whereas a negative result

for the sample DNA of interest and ampliﬁ cation of the IC clearly indicates the absence of

parameter in the sample. The real-time PCR kit minimizes contamination risk and contains

all reagents (except for template DNA) needed for the detection of target DNA. Primers

and probes provide speciﬁ c detection of target DNA in food and environmental samples,

including primary production stage samples. The described performance of the kit is

guaranteed for use only on the real-time PCR instruments listed above.

Step-by-Step Procedure

Using the kit‘s sequence-speciﬁ c primers in a polymerase chain reaction (PCR), the

PCR instrument and the supplied reagents amplify fragments of speciﬁ c sequences for

target DNA.

The PCR instrument detects these ampliﬁ ed fragments in real time through

ﬂ uorescence generated by cleavage of the hybridized probe due to the 5´-nuclease

activity of the Taq DNA polymerase. The probe is labeled at the 5´-end with a reporter

ﬂ uorophore and at the 3´-end with a quencher.

During the annealing/elongation phase of each PCR cycle, the probe hybridizes to

an internal sequence of the amplicon and is cleaved by the 5’ nuclease activity of the

Taq DNA polymerase. This cleavage of the probe separates the reporter dye from the

quencher dye, increasing the reporter dye signal.

The PCR instrument measures the emitted ﬂ uorescence of the reporter dye.

Prevention of Carry-Over Contamination

The heat-labile Uracil-DNA N-Glycosylase (UNG) is suitable for preventing carry-over

contamination between PCR’s. This technique relies on the incorporation of deoxyuridine

triphosphate (dUTP) during all ampliﬁ cation reactions, and the pretreatment of all successive

PCR mixtures with the heat-labile UNG. The UNG cleaves DNA at any site where a

deoxyuridine residue has been incorporated. The resulting abasic sites are hydrolyzed

due to the high temperatures during the initial denaturation step, and can no longer serve

as PCR templates. The heat-labile UNG is inactivated during the initial denaturation step.

Native DNA (e.g., the isolated target genomic DNA) does not contain uracil and is therefore

not degraded by this procedure. Since dTTP is replaced with dUTP and UNG is included in

this kit, decontamination can be achieved with the provided reagents.

KIT 2300 48 - Listeria monocytogenes Detection Kit

ADDITIONAL INFORMATION

3.3 Reference Number

The reference number and original BIOTECON Diagnostics article number: R 302 23.

3.4 Change Index

Version 5, February 2022:

Rebranding, new document layout and updated content.

R 302 23 20 (13)

3.2 Trademarks

foodproof®, microproof®, vetproof®, ShortPrep®, RoboPrep®, and LyoKit® are trademarks

of BIOTECON Diagnostics GmbH.

Hygiena

TM is a registered trademark of Hygiena.

Other brand or product names are trademarks of their respective holders.

Manufactured by

BIOTECON

Diagnostics GmbH

Hermannswerder 17

14473 Potsdam

Germany

[email protected]

www.bc-diagnostics.com

Hygiena LLC

CA 93012 Camarillo

USA

www.hygiena.com/technical-support-request

Hygiena KIT 2300 48 User manual

Hygiena KIT 2300 48 User manual

Hygiena KIT 2300 48 User manual

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